Methods and materials for treating calcific aortic valve stenosis

ABSTRACT

This document provides methods and materials involved in treating cardiovascular conditions such as calcific aortic valve stenosis. For example, methods and materials for using sGC agonists or a combination of sGC agonists and PDE5A inhibitors to reduce calcification of heart valves and/or vessels or to slow progression of aortic sclerosis to calcific aortic valve stenosis are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/269,628, filed Feb. 7, 2019 (now U.S. Pat. No. 10,568,895), which isa continuation of U.S. application Ser. No. 15/702,461, filed Sep. 12,2017 (now U.S. Pat. No. 10,238,669), which is a divisional of U.S.application Ser. No. 14/652,903, filed Jun. 17, 2015 (now U.S. Pat. No.9,789,126), which application is a National Stage application under 35U.S.C. § 371 of International No. PCT/US2013/077254, filed Dec. 20,2013, which application claims the benefit of U.S. ProvisionalApplication Ser. No. 61/740,680, filed Dec. 21, 2012. The disclosures ofthe prior applications are considered part of (and are incorporated byreference in) the disclosure of this application.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant HL092235awarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND 1. Technical Field

This document relates to methods and materials involved in treatingcardiovascular conditions such as calcific aortic valve stenosis. Forexample, this document provides methods and materials for using solubleguanylate cyclase (sGC) agonists (e.g., oxidized or non-oxidized sGCagonists) or a combination of sGC agonists and cGMP-specificphosphodiesterase 5A (PDE5A) inhibitors to reduce calcification of heartvalves and/or vessels or to slow progression of aortic sclerosis tocalcific aortic valve stenosis.

2. Background Information

Calcific aortic valve stenosis is a disease where the opening of theaortic valve is narrowed. The symptoms of calcific aortic valve stenosisvary depending on the degree of valve stenosis. Patients with mild tomoderate calcific aortic valve stenosis may lack symptoms as symptomstypically appear in those patients with severe calcific aortic valvestenosis. Symptoms can include progressive shortness of breath onexertion, syncope, chest pain, and sudden death.

SUMMARY

This document provides methods and materials involved in treatingcardiovascular conditions such as calcific aortic valve stenosis. Forexample, this document provides methods and materials for using sGCagonists or a combination of sGC agonists and PDE5A inhibitors to reducecalcification of heart valves and/or vessels or to slow progression ofaortic sclerosis to calcific aortic valve stenosis. As described herein,sGC agonists such as Ataciguat can be used to slow progression of aorticsclerosis to calcific aortic valve stenosis. Having the ability to slowprogression of aortic sclerosis to calcific aortic valve stenosis canallow patients to live longer and happier lives.

In general, one aspect of this document features a method for slowingprogression of calcification of a heart valve or vessel. The methodcomprises, or consists essentially of, (a) identifying a mammal as beingat risk for heart valve calcification or vessel calcification, and (b)administering a sGC agonist to the mammal, thereby slowing progressionof calcification of a heart valve or vessel within the mammal. Themammal can be a human. The sGC agonist can be selected from the groupconsisting of YC-I, BAY 58-2667, BAY 41-2272, BAY-41-8543, BAY 63-2521,and HMR1766. The method can comprise administering a PDE5A inhibitor tothe mammal.

In another aspect, this document features a method for slowingprogression of aortic sclerosis to calcific aortic valve stenosis. Themethod comprises, or consists essentially of, (a) identifying a mammalhaving aortic sclerosis, and (b) administering a sGC agonist to themammal, thereby slowing progression of aortic sclerosis to calcificaortic valve stenosis within the mammal. The mammal can be a human. ThesGC agonist can be selected from the group consisting of YC-I, BAY58-2667, BAY 41-2272, BAY-41-8543, BAY 63-2521, and HMR1766. The methodcan comprise administering a PDE5A inhibitor to the mammal.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a graph plotting the time course of development of calcificaortic valve stenosis in ldlr^(−/−)/apoB^(100/100) mice.

FIGS. 2A-C contain results demonstrating the effects of sGC activationand NO signaling on osteogenic signaling and progression of aortic valvedysfunction in vitro and in vivo. FIG. 2A demonstrates the effect ofAtaciguat on pVASP239 levels in aortic valve interstitial cells invitro, and FIG. 2B demonstrates the effect of Ataciguat on BMP2-inducedSp7 expression in aortic valve interstitial cells in vitro. FIG. 2Cdemonstrates the effects of DDAH1 overexpression on aortic valvefunction in ldlr-deficient, apoB100-only mice. Overexpression of DDAH1reduces endogenous inhibitors of NOS, increases NO production, andincreases sGC signaling, ultimately resulting in slower progression ofCAVS.

FIGS. 3A-B contain results demonstrating the effects of catalaseoverexpression or deletion (which would increase or decrease sGCoxidation, respectively) on pro-osteogenic gene expression and aorticvalve function in mice. Overexpression of catalase reduces expression ofosteocalcin (a pro-osteogenic gene, FIG. 3A) and slows progression ofCAVS (open circles, FIG. 3B). Deletion of catalase accelerates valvedisease (open squares, FIG. 3B).

FIG. 4 is a graph plotting p-VASP²³⁹ levels detected in aortic valveinterstitial cells treated with BMP2 (100 ng/mL), TGFβ1 (10 ng/mL), orneither (control; CTRL) in the presence or absence of Ataciguat (1 μmol)for 18 hours at 37° C. Ataciguat (HMR1766) effectively activated sGCsignaling in aortic valve interstitial cells.

FIG. 5 is a graph plotting p-Smad1/5/8 levels detected in aortic valveinterstitial cells treated with BMP2 (100 ng/mL), TGFβ1 (10 ng/mL), orneither (control; CTRL) in the presence or absence of Ataciguat (1 μmol)for 18 hours at 37° C. Ataciguat (HMR1766) attenuated canonical BMP2signaling in aortic valve interstitial cells.

FIG. 6 is a graph plotting Sp7 levels detected in aortic valveinterstitial cells treated with BMP2 (100 μg/mL), TGFβ1 (10 μg/mL), orneither (control; CTRL) in the presence or absence of Ataciguat (1 μmol)for 18 hours at 37° C. Ataciguat (HMR1766) attenuated BMP targetinduction in aortic valve interstitial cells.

FIG. 7 is a graph plotting p-Smad2 levels detected in aortic valveinterstitial cells treated with BMP2 (100 μg/mL), TGFβ1 (10 μg/mL), orneither (control; CTRL) in the presence or absence of Ataciguat (1 μmol)for 18 hours at 37° C. Ataciguat (HMR1766) partially attenuatedfibrogenic signaling in aortic valve interstitial cells.

DETAILED DESCRIPTION

This document provides methods and materials involved in treatingcardiovascular conditions such as calcific aortic valve stenosis. Forexample, this document provides methods and materials for using one ormore sGC agonists or a combination of one or more sGC agonists and oneor more PDE5A inhibitors to reduce calcification of heart valves and/orvessels or to slow progression of aortic sclerosis to calcific aorticvalve stenosis.

Any type of mammal having a cardiovascular condition such as calcificaortic valve stenosis can be treated as described herein. For example,humans and other primates such as monkeys having a cardiovascularcondition such as calcific aortic valve stenosis can be treated with oneor more sGC agonists or a combination of one or more sGC agonists andone or more PDE5A inhibitors. In some cases, dogs, cats, horses, cows,pigs, sheep, mice, and rats can be treated with one or more sGC agonistsor a combination of one or more sGC agonists and one or more PDE5Ainhibitors as described herein.

Any appropriate method can be used to identify a mammal having acardiovascular condition such as calcific aortic valve stenosis oraortic sclerosis. For example, echocardiography or computed tomographyscanning can be used to identify a human having aortic sclerosis that isat risk of progressing into calcific aortic valve stenosis.

Once identified as having a cardiovascular condition such as calcificaortic valve stenosis or aortic sclerosis with the potential to progressinto calcific aortic valve stenosis, the mammal can be administered orinstructed to self-administer one or more sGC agonists or a combinationof one or more sGC agonists and one or more PDE5A inhibitors. Examplesof sGC agonists include, without limitation, Ataciguat, YC-I, BAY58-2667, BAY 41-2272, and BAY-41-8543. Examples of PDE5A inhibitorsinclude, without limitation, sildenafil, vardenafil, tadalafil, EMD360527, DA 8159, UK-343-664 (Walker et al., Xenobiotica, 31:651-664(2001)), UK-427-387, UK-357903([1-ethyl-4-{3-[3-ethyl-6,7-dihydro-7-oxo-2-(2-pyridylmethyl)-2H-pyrazolo[4,3-d]pyrimidin-5-yl]-2-(2-methoxyethoxy)-5-pyridylsulphonyl}piperazine])(Gardiner et al., J. Pharmacol. Exp. Ther., 312:265-271 (2005)),UK-371800 (Pfizer), UK-313794 (Pfizer), UK-343664 (Abel et al.,Xenobiotica, 31:665-76 (2001)), TA-1790 (Tanabe Seiyaku), CP-248 (OsiPharmaceuticals), CP-461 (Osi Pharmaceuticals), exisulind (Deguchi etal., Molecular Cancer Therapeutics, 803-809 (2002); (OsiPharmaceuticals)), pyrazolinone, EMD82639 (Merck KgaA, Darmstadt, DE;(4-(4-[2-ethyl-phenylamino)-methylene]-3-methyl-5-oxo-4,5-di-hydro-pyrazol-1-yl)-benzoicacid; Senzaki et al., FASEB J., 15:1718-1726 (2001), and Scutt et al.,BMC Pharmacol., 4:10 (2004)), EMD360527 (Merck KgaA, Darmstadt, DE;[7-(3-Chloro-4-methoxy-benzylamino)-1-methyl-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-5-ylmethoxy]-aceticacid; Scutt et al., BMC Pharmacol., 4:10 (2004)), EMD221829 (Merck KgaA,Darmstadt, DE;4-[4-(3-Chloro-4-methoxy-benzylamino)-benzo[4,5]thieno[2,3-d]-pyrimidin-2-yl]-cyclohexanecarboxylicacid, ethanolamin salt; Scutt et al., BMC Pharmacol., 4:10 (2004)), EMD171827 (Merck KgaA, Darmstadt, DE;5-[4-(3-Chloro-4-methoxy-benzylamino)-5,6,7,8-tetrahydro-benzo[4,5]thieno[2,3-d]pyrimidin-2-yl]-pentanoicacid; Scutt et al., BMC Pharmacol., 4:10 (2004)), DA-8259(3-(1-Methyl-7-oxo-3-propyl-6,7-dihydro-1H-pyrazolo-[4,3-d]pyrimidin-5-yl)-N-[2-(1-methylpyrrolidin-2-yl)ethyl]-4-propoxybenzenesulfonamide),E-4021 (Dukarm et al., Am. J. Respir. Crit. Care Med., 160:858-865(1999)), pentoxifylline, and FR22934 (Fujisawa). Additional examples ofPDE5A inhibitors can be set forth in U.S. Pat. Nos. 6,916,927,6,911,542, 6,903,099, 6,878,711, 6,872,721, 6,858,620, 6,825,197,6,774,128, 6,723,719, 6,699,870, 6,670,366, 5,859,006, and 5,250,534 andInternational Patent Application Publication No. WO 03/063875, WO03/1012761, WO 2004/037183, and WO 98/38168. In some cases, a sGCagonist used as described herein can be an activator of an oxidized ornon-oxidized form of sGC.

In some cases, one or more sGC agonists (e.g., one, two, three, four,five, or more sGC agonists) or a combination of one or more sGC agonists(e.g., one, two, three, four, five, or more sGC agonists) and one ormore PDE5A inhibitors (e.g., one, two, three, four, five, or more PDE5Ainhibitors) can be administered to a mammal to reduce calcification ofheart valves and/or vessels or to slow progression of aortic sclerosisto calcific aortic valve stenosis. In some cases, one or more sGCagonists or a combination of one or more sGC agonists and one or morePDE5A inhibitors can be formulated into a pharmaceutically acceptablecomposition. For example, a therapeutically effective amount ofAtaciguat can be formulated together with one or more pharmaceuticallyacceptable carriers (additives) and/or diluents. A pharmaceuticalcomposition can be formulated for administration in solid or liquid formincluding, without limitation, sterile solutions, suspensions,sustained-release formulations, tablets, capsules, pills, powders, andgranules.

Pharmaceutically acceptable carriers, adjuvants and vehicles that may beused in the pharmaceutical compositions described herein include,without limitation, ion exchangers, alumina, aluminum stearate,lecithin, serum proteins, such as human serum albumin, buffer substancessuch as phosphates, glycine, sorbic acid, potassium sorbate, partialglyceride mixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,polyethylene glycol and wool fat. If required, the solubility andbioavailability of a sGC agonist and/or a PDE5A inhibitor in apharmaceutical composition can be enhanced using lipid excipients and/orblock copolymers of ethylene oxide and propylene oxide. See, e.g., U.S.Pat. No. 7,014,866 and U.S. Patent Publication Nos. 20060094744 and20060079502.

A pharmaceutical composition described herein can be designed for oralor parenteral (including subcutaneous, intramuscular, intravenous, andintradermal) administration. Compositions suitable for parenteraladministration include aqueous and non-aqueous sterile injectionsolutions that can contain anti-oxidants, buffers, bacteriostats, andsolutes which render the formulation isotonic with the blood of theintended recipient; and aqueous and non-aqueous sterile suspensionswhich may include suspending agents and thickening agents. Theformulations can be presented in unit-dose or multi-dose containers, forexample, sealed ampules and vials, and may be stored in a freeze dried(lyophilized) condition requiring only the addition of the sterileliquid carrier, for example water for injections, immediately prior touse. Extemporaneous injection solutions and suspensions may be preparedfrom sterile powders, granules, and tablets.

Such injection solutions can be in the form, for example, of a sterileinjectable aqueous or oleaginous suspension. This suspension may beformulated according to techniques known in the art using suitabledispersing or wetting agents (such as, for example, Tween 80) andsuspending agents. The sterile injectable preparation can be a sterileinjectable solution or suspension in a non-toxic parenterally-acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that can be used are mannitol,water, Ringer's solution, and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose, any bland fixed oil can be usedincluding synthetic mono- or di-glycerides. Fatty acids, such as oleicacid and its glyceride derivatives can be used in the preparation ofinjectables, as can natural pharmaceutically-acceptable oils, such asolive oil or castor oil, including those in their polyoxyethylatedversions. These oil solutions or suspensions can contain a long-chainalcohol diluent or dispersant.

In some cases, a pharmaceutically acceptable composition including oneor more sGC agonists and/or one or more PDE5A inhibitors can beadministered locally or systemically. For example, a compositioncontaining a sGC agonist can be administered systemically by injectionto a mammal (e.g., a human). In some cases, each sGC agonist to beadministered when two or more sGC agonists are to be administered can beadministered by the same or different routes. For example, Ataciguat canbe administered orally, and YC-I can be administered by injection. Insome cases, one or more sGC agonists can be administered via one route,and one or more PDE5A inhibitors can be administered via the same or adifferent route.

A composition containing one or more sGC agonists or a combination ofone or more sGC agonists and one or more PDE5A inhibitors can beadministered to a mammal in any amount, at any frequency, and for anyduration effective to achieve a desired outcome (e.g., to reducecalcification of heart valves and/or vessels or to slow progression ofaortic sclerosis to calcific aortic valve stenosis).

Effective doses can vary, as recognized by those skilled in the art,depending on the severity of the condition (e.g., calcific aortic valvestenosis), the route of administration, the sex, age and general healthcondition of the subject, excipient usage, the possibility of co-usagewith other therapeutic treatments such as use of other agents and thejudgment of the treating physician.

An effective amount of a composition containing one or more sGC agonistsor a combination of one or more sGC agonists and one or more PDE5Ainhibitors can be any amount that reduces the severity of a symptom of acondition being treated (e.g., calcific aortic valve stenosis) withoutproducing significant toxicity to the mammal. For example, an effectiveamount of a sGC agonist such as YC-I can be from about 0.5 mg/kg toabout 80 mg/kg (e.g., from about 0.5 mg/kg to about 70 mg/kg, from about0.5 mg/kg to about 60 mg/kg, from about 0.5 mg/kg to about 50 mg/kg,from about 0.5 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30mg/kg, from about 0.5 mg/kg to about 20 mg/kg, from about 0.5 mg/kg toabout 10 mg/kg, from about 0.5 mg/kg to about 5 mg/kg, from about 0.5mg/kg to about 1 mg/kg, from about 0.75 mg/kg to about 10 mg/kg, fromabout 1 mg/kg to about 10 mg/kg, or from about 2 mg/kg to about 10mg/kg). In some cases, between about 50 mg and 200 mg (e.g., betweenabout 50 mg and 180 mg, between about 50 mg and 150 mg, between about 50mg and 125 mg, between about 60 mg and 200 mg, between about 75 mg and200 mg, between about 100 mg and 200 mg, between about 75 mg and 150 mg,or between about 100 mg and 150 mg) of a sGC agonist such as Ataciguatcan be administered to an average sized human (e.g., about 70 kg human)daily for about 20 weeks. If a particular mammal fails to respond to aparticular amount, then the amount of sGC agonist or PDE5A inhibitor canbe increased by, for example, two fold. After receiving this higheramount, the mammal can be monitored for both responsiveness to thetreatment and toxicity symptoms, and adjustments made accordingly. Theeffective amount can remain constant or can be adjusted as a slidingscale or variable dose depending on the mammal's response to treatment.Various factors can influence the actual effective amount used for aparticular application. For example, the frequency of administration,duration of treatment, use of multiple treatment agents, route ofadministration, and severity of the condition (e.g., calcific aorticvalve stenosis) may require an increase or decrease in the actualeffective amount administered.

The frequency of administration can be any frequency that reduces theseverity of a symptom of a condition to be treated (e.g., calcificaortic valve stenosis) without producing significant toxicity to themammal. For example, the frequency of administration can be from aboutonce a week to about three times a day, or from about twice a month toabout six times a day, or from about twice a week to about once a day.The frequency of administration can remain constant or can be variableduring the duration of treatment. A course of treatment with acomposition containing one or more sGC agonists or a combination of oneor more sGC agonists and one or more PDE5A inhibitors can include restperiods. For example, a composition containing one or more sGC agonistsor a combination of one or more sGC agonists and one or more PDE5Ainhibitors can be administered daily over a two week period followed bya two week rest period, and such a regimen can be repeated multipletimes. As with the effective amount, various factors can influence theactual frequency of administration used for a particular application.For example, the effective amount, duration of treatment, use ofmultiple treatment agents, route of administration, and severity of thecondition (e.g., calcific aortic valve stenosis) may require an increaseor decrease in administration frequency. An effective duration foradministering a composition containing one or more sGC agonists or acombination of one or more sGC agonists and one or more PDE5A inhibitorscan be any duration that reduces the severity of a symptom of thecondition to be treated (e.g., calcific aortic valve stenosis) withoutproducing significant toxicity to the mammal. Thus, the effectiveduration can vary from several days to several weeks, months, or years.In general, the effective duration for the treatment of calcific aorticvalve stenosis can range in duration from several months to severalyears. In some cases, an effective duration can be for as long as anindividual mammal is alive. Multiple factors can influence the actualeffective duration used for a particular treatment. For example, aneffective duration can vary with the frequency of administration,effective amount, use of multiple treatment agents, route ofadministration, and severity of the condition being treated.

In certain instances, a course of treatment and the severity of one ormore symptoms related to the condition being treated can be monitored.Any appropriate method can be used to determine whether or not theseverity of a symptom is reduced. For example, the severity of a symptomof calcific aortic valve stenosis can be assessed using imagingtechniques at different time points.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1—LDLR^(−/−)/apoB^(100/100) Mice Emulate Human CalcificAortic Valve Stenosis

LDLR^(−/−)/apoB^(100/100) mice are low density lipoproteinreceptor-deficient, apolipoprotein B100-only mice. They were found todevelop severe, hemodynamically significant calcific aortic valvestenosis consistently. The peak velocity was about 4 m/sec (FIG. 1).These results indicate that these mice develop stenosis in a mannersuitable for studying the effects of pharmacological interventions oninitiation and progression of calcific aortic valve stenosis.

Example 2—Soluble Guanylate Cyclase Activators Increase NO Signalingwhich can Slow Progression of Calcific Aortic Valve Stenosis

To determine whether induction of sGC signaling reduced osteogenicsignaling in vitro, aortic valve interstitial cells were treated withBMP2 in the presence or absence of Ataciguat (HMR1766,5-Chloro-2-[[(5-chloro-2-thienyl)sulfonyl]amino]-N-[4-(4-morpholinylsulfonyl)phenyl]benzamide)for 24 hours. Ataciguat activated sGC signaling (indicated by pVASP239levels) and profoundly suppressed osteogenic signaling in aortic valveinterstitial cells in vitro (FIGS. 2A and 2B).

To determine whether increasing NO production and sGC signaling can slowprogression of calcific aortic valve stenosis in vivo, the effects ofoverexpressing human DDAH1 (DDAH1TG, an enzyme that degrades endogenousinhibitors of NOS) on aortic valve function in ldlr-deficient,apoB100-only mice were examined. Increasing NO production and sGCsignaling in DDAHTG mice slowed progression of calcific aortic valvestenosis (FIG. 2C).

These results demonstrate that soluble guanylate cyclase activators suchas Ataciguat activate soluble guanylate cyclase in aortic valveinterstitial cells and that increasing NO signaling slows progression ofcalcific aortic valve stenosis in mammals.

Additional experiments were performed to confirm that Ataciguatactivates sGC signaling, attenuates BMP2 signaling, attenuates BMP2target induction, and attenuates fibrogenic signaling in valveinterstitial cells. In brief, cells were maintained in Dulbecco'sModified Eagle's Medium (DMEM) with 20% fetal bovine serum (FBS) andexpanded to confluence in 6 well plates in a standard 5% CO₂ incubatorat 37° C. Cells were then treated with BMP2 (100 ng/mL), TGFβ1 (10ng/mL), or vehicle (phosphate buffered saline) in the absence orpresence of Ataciguat (1 μmol) for 18 hours. Cells were lysed usingstandard protein lysis buffer containing protease and phosphataseinhibitors, and Western blotting was performed to evaluate changes inprotein levels of BMP, TGFβ, and sGC signaling.

Ataciguat activated sGC signaling (p-VASP²³⁹ levels, FIG. 4), attenuatedBMP2 signaling (p-Smad1/5/8, FIG. 5), attenuated BMP2 target induction(Sp7, FIG. 6), and partially attenuated fibrogenic signaling (p-Smad2,FIG. 7) in valve interstitial cells. These results were replicated inhuman cells lines (HAV-001, HAV-002, HAV-003) and mouse cell lines(MAV1, MAV2, and MAV4).

Example 3—Reducing Oxidative Stress Slows Progression of Calcific AorticValve Stenosis

To determine whether hydrogen peroxide levels are associated withaltered osteogenic gene expression and aortic valve dysfunction,ldlr-deficient, apoB100-only mice that either overexpress human catalaseor are deficient in catalase were generated. Overexpression of catalase,which reduces oxidative stress, reduced soluble guanylate cyclaseoxidation, increased NO signaling, reduced expression of pro-osteogenicgenes, and slowed progression of calcific aortic valve stenosis (FIGS.3A and 3B). Deletion of catalase, which increases oxidative stress,increased sGC oxidation, reduced NO signaling, and acceleratedprogression of calcific aortic valve stenosis in hypercholesterolemicmice (FIG. 3B).

These results indicate that increases in oxidative stress contribute tosoluble guanylate cyclase oxidation, pro-osteogenic gene expression, andprogression of calcific aortic valve stenosis in hypercholesterolemicmice.

Example 4—Reducing Pro-Osteogenic Signaling and Slowing Progression ofCalcific Aortic Valve Stenosis

To confirm that administration of soluble guanylate cyclase activators(e.g., Ataciguat) reduces pro-osteogenic signaling and slows progressionof calcific aortic valve stenosis in mammals, ldlr^(−/−)/apoB^(100/100)mice are maintained on a Western diet for 6 months, and then are placedinto one of four groups: 1) Vehicle, 2) 5 mg/kg Ataciguat/day, or 3) 10mg/kg Ataciguat/day, or 4) 15 mg/kg Ataciguat/day for an additional 3months. Vehicle or Ataciguat is administered daily by oral gavage.Changes in cardiac and aortic valve function are evaluated at 3, 6, and9 months in all four groups of mice. 20 mice of each treatment group aresacrificed at the 9 month time point, which typically represents a stageof moderate to severe calcific aortic valve stenosis in otherwiseuntreated mice. One group of 10 mice is used for semi-quantitativehistological/immunohistochemical evaluation of aortic valve (calcium,osteogenic signaling molecules; n=10). The second group is used formeasurement of pro-osteogenic and pro-fibrotic gene expression usingqRT-PCR (n=10).

To confirm that soluble guanylate cyclase activators (e.g., Ataciguat)effectively attenuates responses to specific signals that induceosteogenesis in aortic valve interstitial cells, transcriptionalresponses to various pro-osteogenic molecules in the presence andabsence of exogenous oxidative stress (which induce soluble guanylatecyclase oxidation) are examined. In brief, responses to bonemorphogenetic protein 2, bone morphogenetic protein 4, and transforminggrowth factor β-1 (all with or without 100 μM H₂O₂) are examined for theability to be altered by Ataciguat (1 μM). These stimuli are known to beincreased in human calcific aortic valve stenosis.

To confirm tolerance of Ataciguat in humans with calcific aortic valvestenosis, patients receiving Ataciguat are monitored. In brief, subjectswith asymptomatic, mild-to-moderate calcific aortic valve stenosis areadministered placebo, 2.5 mg/kg, 5 mg/kg, or 10 mg/kg Ataciguat (orally)on separate visits and are monitored for 12 hours. Blood pressure ismonitored non-invasively and continuously using Dinamap™ and Finapres™equipment. Every 2 hours, subjects are asked to stand up from sitting orlying down to determine orthostatic tolerance. 40 patients are recruitedfor this study.

To confirm that Ataciguat can be used to treat calcific aortic valvestenosis, humans with severe calcific aortic valve stenosis areadministered placebo or the highest tolerated dose of Ataciguat 24 hoursprior to aortic valve replacement surgery. Patients are admitted to thehospital the preceding day to ensure continuous monitoring followingthis dose. When valve tissue is excised during surgery, a studycoordinator takes two small portions of a calcified valve cusp. One isembedded in OCT for cryosectioning, and the second is snap frozen inliquid nitrogen. OCT-embedded tissue is used to determine spatialchanges in soluble guanylate cyclase, p-VASP239, and osteogenic proteinlevels. The second piece is pulverized and is used for quantitativeexamination of gene expression (qRT-PCR) and protein levels forpro-osteogenic molecules and molecules related to NO-soluble guanylatecyclase signaling.

A randomized, double-blinded study is designed with patients diagnosedwith mild to moderate calcific aortic valve stenosis. Patients receiveeither placebo or Ataciguat. Prior to enrollment in the trial, patientsundergo routine, clinically-indicated evaluations of aortic valve andventricular function using echocardiography and aortic valve calciumusing CT imaging. Patients also have blood drawn for evaluation ofchanges in inflammatory cytokines and circulating p-VASP239 levels.Following 1 month of run-in treatment, patients return for evaluation oftolerance and humoral efficacy (i.e., increases in circulating p-VASP239levels in treated groups). Aortic valve function and aortic valvecalcium burden are evaluated every 6 months by echocardiography and CTimaging, respectively.

Example 5—Using Soluble Guanylate Cyclase Agonists to Slow Progressionof Calcification of Heart Valves and Vessels

A patient is identified as having aortic valve or vascular calcificationthrough regular echocardiographic screening (typical for a physical examat age 65 or greater) or following computed tomography scanning of thechest. The patient with evidence of cardiovascular calcification(usually bright, echogenic areas on the valve/vessels) is treated withoral Ataciguat on a daily basis (50 mg/day, 100 mg/day, or 200 mg/day)to slow progression of cardiovascular calcification. P-VASP239phosphorylation levels in peripheral blood are used to monitor drugbioavailability following 1-2 weeks of treatment. The patient undergosfollow-up testing at 1 year with computed tomography scanning toevaluate quantitatively progression of heart valve or vesselcalcification. Oral Ataciguat treatment proceeds for the remainder of asubject's lifetime, so long as it is well-tolerated.

Example 6—Using a Combination of Soluble Guanylate Cyclase Agonists andcGMP-Specific Phosphodiesterase 5A Inhibitors to Slow Progression ofCalcification of Heart Valves and Vessels

A patient is identified as having aortic valve or vascular calcificationthrough regular echocardiographic screening (typical for a physical examat age 65 or greater) or following computed tomography scanning of thechest. The patient with evidence of cardiovascular calcification(usually bright, echogenic areas on the valve/vessels) is treated with acombination of oral Ataciguat (50 mg/day, 100 mg/day, or 200 mg/day) anda low dose of a PDE5A inhibitor (e.g., Sildenafil or Tadalafil, 2.5mg/day, 5 mg/day, or 10 mg/day) to slow progression of cardiovascularcalcification. P-VASP239 phosphorylation levels in peripheral blood areused to monitor drug bioavailability following 1-2 weeks of treatment.The subject undergoes follow-up testing at 1 year with computedtomography scanning to evaluate quantitatively progression of heartvalve or vessel calcification. Treatment with an oral sGCagonist/PDE5Ainhibitor combination proceeds for the remainder of a subject'slifetime, as long as it is well-tolerated.

Example 7—Using Soluble Guanylate Cyclase Agonists to Slow Progressionof Aortic Sclerosis to Calcific Aortic Valve Stenosis

A patient is identified as having aortic valve sclerosis (or mild aorticvalve stenosis) through regular echocardiographic screening (typical fora physical exam at age 65 or greater). The patient with evidence ofcardiovascular calcification (usually bright, echogenic areas on thevalve) is treated with oral sGC activators/stimulators (50 mg/day, 100mg/day, or 200 mg/day) to slow progression of aortic valve calcificationand fibrosis, which can also slow progression of aortic valvedysfunction and progression to aortic valve stenosis. P-VASP239phosphorylation levels in peripheral blood are used to monitor drugbioavailability following 1-2 weeks of treatment. The subject undergoesfollow-up testing at 1 year with echocardiographic imaging to evaluatequantitatively progression of heart valve dysfunction (i.e., reductionsin aortic valve area or increases in transvalvular velocity). Treatmentwith an oral sGC agonist proceeds for the remainder of a subject'slifetime, as long as it is well-tolerated.

Example 8—Using a Combination of Soluble Guanylate Cyclase Agonists andcGMP-Specific Phosphodiesterase 5A Inhibitors to Slow Progression ofAortic Sclerosis to Calcific Aortic Valve Stenosis

A patient is identified as having aortic valve sclerosis (or mild aorticvalve stenosis) through regular echocardiographic screening (typical fora physical exam at age 65 or greater). The patient with evidence ofcardiovascular calcification (usually bright, echogenic areas on thevalve/vessels) is treated with a combination of oral Ataciguat (50mg/day, 100 mg/day, or 200 mg/day) and a low dose of a PDE5A inhibitor(e.g., Sildenafil or Tadalafil, 2.5 mg/day, 5 mg/day, or 10 mg/day) toslow progression of aortic valve calcification and fibrosis, which canalso slow progression of aortic valve dysfunction and progression toaortic valve stenosis. P-VASP239 phosphorylation levels in peripheralblood are used to monitor drug bioavailability following 1-2 weeks oftreatment. The subject undergoes follow-up testing at 1 year withechocardiographic imaging to evaluate quantitatively progression ofheart valve dysfunction (i.e., reductions in aortic valve area orincreases in transvalvular velocity). Treatment with an oral sGCagonist/PDE5A inhibitor combination proceeds for the remainder of asubject's lifetime, as long as it is well-tolerated.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method for slowing progression of calcificationof a heart valve, wherein said method comprises administering a sGCagonist, as the sole active ingredient for slowing said progression, toa mammal identified as having heart valve calcification, thereby slowingprogression of calcification of a heart valve within said mammal,wherein the sGC agonist is YC-I (3-(5′-hydroxymethyl-2′furyl)-1-benzylindazole) or HMR-1766(5-Chloro-2-[[5-chloro-2-thienyl)sulfonyl]amino]-N-[4-(4-morpholinylsulfonyl)phenyl]benzamide).2. The method of claim 1, wherein said mammal is a human.
 3. The methodof claim 1, wherein said sGC agonist is YC-I.
 4. The method of claim 1,wherein said sGC agonist is HMR-1766.